Most important biological and biomedical processes are carried out not by isolated macromolecules but by complexes of interacting macromolecules. We are studying the structures of several such assemblies and the interactions among their components, using electron microscopy in combination with other approaches. The complexes currently under study are important for: amyloidosis, with particular reference to the connection between amyloid formation and prion biology; intracellular proteolyis of defective and regulatory proteins; and the structure and assembly of clathrin-coated vesicles.[unreadable] [unreadable] (1) Amyloid Filament Formation by the Fungal Prions, Ure2p, Sup35p, and Het-s, and Other Amyloidogenic Proteins. Amyloid is fibrous aggregates of protein(s) in protease-resistant, beta-sheet-rich, non-native conformations that accumulate in certain disease-related situations, including rheumatoid arthritis. Prions (infectious proteins) are transmissible amyloids that have been implicated in neuropathies, including the spongiform encephalopathies. To investigate amyloids and the mechanisms that underlie their formation, we started studying the structures of yeast prions in 1998. We focussed initially on Ure2p, a protein that normally functions as a negative regulator of nitrogen catabolism. We showed that the N-terminal ?prion domain? of Ure2p is responsible for filament formation, and the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our ?amyloid backbone? concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In FY05, we published the ?parallel superpleated beta-structure model? that we proposed as applicable to Ure2p filaments and several other amyloids. It envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. In FY06, we focused on two areas: (i) electron microscopy of prion domain filaments of Het-S from Podospora anserina, which differs from Ure2p and Sup35p in being a gain-of function prion, not a loss-of-function prion, and in not having a high concentration of Asn/Gln residues. We showed by electron diffraction that Het-s filaments have a cross-beta structure, and performed mass-per-unit-length measurements by scanning transmission electron microscopy. The latter data show that it has an axial packing density of 1 subunit per 0.94 nm ? half that of Ure2p and Sup35p filaments and in agreement with a published model, the stacked beta-solenoid. (ii) We surveyed the structural properties manifested in known crystal structures of beta-helical proteins, which have many features in common with amyloid, and found that their turns ? which we call ?beta-arches? - are systematically different from the beta-turns that are characteristic of conventional globular proteins. This information will be exploited to refine our amyloid models.[unreadable] [unreadable] (2) Role of Energy-dependent Proteases in Protein Quality Control and Cell Regulation. All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute the cell. Programmed degradation of regulatory factors is a major factor in controlling the cell cycle. Both activities are carried out by energy-dependent proteases, which consist of two parts - a peptidase and a chaperone-like ATPase. The archetypal complex of this kind is the proteasome. For several years, our studies focussed on the Clp proteases of E. coli, a model system with a similar repertoire. We showed that the peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpP to form active complexes. We went on to show that substrate proteins bind to distal sites on the ATPase and are then translocated axially into the digestion chamber inside ClpP. In FY06, we continued working to mesh the information in our cryo-EM structures of fully assembled proteases in solution with high resolution structures of individual subunits as packed into crystals. In principle, discrepancies may be indicative of local mobility, differences in nucleotide state, or crystal contact effects. To this end, we compared 1-nm resolution reconstructions of hexamers of full-length ClpA and of a mutant lacking its 16-kDa N-domain and found them to be indistinguishable. This observation substantiated earlier evidence that the N-domains are so mobile as to become invisible after they are averaged in a reconstruction. The discreet sites at which they reside in crystals presumably represent capture by crystal contacts. The N-domain of ClpB and its 9-kDa coiled-coil domain are similarly mobile. We also observed mobility in near-axial regions of the D2 domains of both ClpA and ClpB as well as in ClpQ. This trend and the positioning of the mobile regions suggest that their mobility is exploited in some way in the translocation of unfolded substrates along the central axis of the hexamers. [unreadable] [unreadable] (3) Interaction of Clathrin with Proteins that Regulate its Assembly. Clathrin plays a key role in intracellular trafficking, via its polymerization into the coats of coated pits and vesicles. Assembly of clathrin is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. In the 1980s, we studied the molecular composition of coated vesicles and the plasticity of the assembly unit, the clathrin triskelion. We returned to this system in FY05, equipped with cryo-EM technology, and compared the structures of coated vesicles with and without binding of the uncoating ATPase, Hsc70. On the basis of these observations, we developed a model for uncoating. In FY06, we extended these studies to investigate the pleiomorphy (structural variability) of coated vesicles (CVs), their vesicular contents (or lack thereof), and their complements of internal proteins, i.e. lying between the coat and the vesicle. To this end, we have been performing cryo-electron tomography on CVs isolated from bovine brain. The observed coated particles (CPs) range in diameter from 66 nm to 120 nm, with those equal to or larger than 80 nm having an enclosed vesicle, from 23 nm to 57 nm in diameter. The smaller particles are called clathrin baskets (CBs) because they lack vesicles, while the larger particles represent true CVs. CPs occur in many different forms, and may be described in part by the symmetry of their polygonal facets. Only a subset of all possible polyhedral forms was observed, suggesting that these are the lowest energy forms. The smallest polyhedron found to enclose a vesicle has 38 vertices, indicating that smaller CPs have insufficient volume to accommodate a vesicle. Segmentation of the CVs showed that the volume and density in the interstitial space, the region between the clathrin coat and the vesicle membrane, is consistent with an upper limit to the stoichiometry of about 1.4 adaptor proteins (AP-2 and AP-2) per triskelion.